Methods of Simultaneously Treating Ocular Rosacea and Acne Rosacea

ABSTRACT

A method for simultaneously treating ocular rosacea and acne rosacea in a human in need thereof comprising administering systemically to said human a tetracycline compound in an amount that is effective to treat ocular rosacea and acne rosacea but has substantially no antibiotic activity.

The present application claims benefit of U.S. provisional applicationSer. No. 60/373,141, filed Apr. 16, 2002, which is incorporated hereinby reference.

BACKGROUND OF THE INVENTION

Ocular rosacea is a common tear film and ocular surface disorder causingeye irritation. This disorder is characterized by eye surfaceinflammation, and a variety of related eye disorders such asblepharitis; meibomian gland disease, including meibomian glanddysfunction and meibomianitis; keratitis; conjunctival hyperemia; andeyelid hyperemia.

A conservative estimate of the number of patients affected with ocularrosacea is 10 million in the United States alone. It has been reportedthat 15% of patients with ocular rosacea develop recurrent cornealepithelial erosions, a potentially sight-threatening problem. Theincidence of ocular rosacea increases with age.

Common complaints of patients suffering from ocular rosacea includeblurred or filmy vision, burning or foreign body sensations in the eye,photophobia, and pain severe enough to awaken the person from sleep.Anterior erosion of the mucocutaneous junction of the eyelid is oftennoted, as well as eyelid and conjunctival infection, eyelid marginirregularity, corneal epithelial changes, and corneal vascularization.

Although patients with ocular rosacea usually have a normal productionof aqueous tears by their lacrimal glands, their meibomian glands canatrophy. The meibomian glands are situated upon the inner surface of theeyelids, between the tarsal plates and conjunctiva. The oily secretionsof these glands lubricate the eyelids.

Ocular rosacea is characterized by inflammation of the eyelids, referredto as blepharitis. Blepharitis can be categorized anatomically intoanterior and posterior blepharitis.

Anterior blepharitis refers to inflammation mainly centered around theeyelashes and follicles. Anterior blepharitis usually is subdividedfurther into staphylococcal and seborrheic variants. Frequently, aconsiderable overlap exists in these processes in individual patients.

Posterior blepharitis mainly is related to dysfunction of the meibomianglands. Alterations in secretory metabolism and function lead todisease. The meibomian secretions become more waxlike and begin to blockthe gland orifices. The stagnant material becomes a growth medium forbacteria and can seep into the deeper eyelid tissue layers, causinginflammation. These processes lead to gland plugging, inspissatedmaterial, formation of chalazia and meibomianitis.

Meibomianitis is characterized by inflammation centered about themeibomian glands. The inflammation can lead to meibomian glanddysfunction, which is characterized by the loss of meibomian gland oilfrom the tear film, an increase in tear film evaporation, a loss ofwater from the tear film and the development of dry eye surface disease.

Methods of treating ocular rosacea have included treatment of theapparent infection/inflammation of the eyelids or meibomian glands. Forexample, patients with ocular rosacea have been symptomatically treatedwith artificial tears, or hot compresses which liquefy the secretions ofthe meibomian glands. However, these methods provide limited, if any,improvement. Also, patients have been treated with topically appliedsteroids to the eyelids or ocular surface. However, steroids are notgood long-term solutions because of the potential side-effects e.g.,cataract and glaucoma.

Additionally, orally administered tetracyclines and tetracyclineanalogues (e.g., doxycycline and minocycline) having antibiotic activityare commonly and effectively used for prophylactic or therapeutictreatment of meibomian gland disease. However, a disadvantage of usingsystemically administered antibiotic tetracyclines is that a highpercentage of patients are unable to tolerate oral tetracyclines forextended periods of time. Also, patients can build up a resistance toantibiotic tetracyclines.

Recently other methods for treating ocular rosacea have been disclosed.For example, Gilbard discloses topical antibiotic tetracyclines for thetreatment of ocular rosacea (International Application WO 00/07601).Additionally, Pflugfelder et al. have disclosed the use of systemic andtopical tetracyclines at a sub-antimicrobial dose for the treatment ofocular rosacea (International Application WO 99/58131).

The skin disease acne rosacea often accompanies ocular rosacea. Inparticular, ocular rosacea is present in approximately 60% ofindividuals with acne rosacea.

Acne rosacea is characterized by inflammatory lesions and permanentdilation of blood vessels. Acne rosacea can also include papules,pustules, and hypertrophic sebaceous glands in facial flush areas. Amanifestation of severe acne rosacea is rhinophyma. Rhinophyma is seenmore often in men with acne rosacea than in women. Rhinophyma ischaracterized by a thickened, lobulated overgrowth of the sebaceousglands and epithelial connective tissue of the nose.

It is well known that acne and ocular rosacea commonly occur together.Nevertheless, no one has disclosed the simultaneous treatment of bothdisorders.

Since many patients are susceptible to the simultaneous occurrence ofboth acne rosacea and ocular rosacea, there is a need for a method ofsimultaneously treating a patient suffering from both types ofdisorders. It is especially advantageous if a single agent would beeffective to treat both types of disorders. The use of a single agentwould reduce both the cost and risk of side effects of treatment.

SUMMARY OF INVENTION

The present invention provides a method for simultaneously treatingocular rosacea and acne rosacea in a human in need thereof. The methodcomprises administering systemically to the human a tetracyclinecompound in an amount that is effective to treat ocular rosacea and acnerosacea but has substantially no antibiotic activity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the photoirritancy factor (PIF) for some tetracyclinecompounds. For structure K, the compounds indicated are as follows:

COL R7 R8 R9 308 hydrogen hydrogen amino 311 hydrogen hydrogenpalmitamide 306 hydrogen hydrogen dimethylaminoFor structures L, M, N or O the compounds indicated are as follows:

COL R7 R8 R9 801 hydrogen hydrogen acetamido 802 hydrogen hydrogendimethylaminoacetamido 804 hydrogen hydrogen nitro 805 hydrogen hydrogenaminoFor structure P, R8 is hydrogen and R9 is nitro (COL-1002).

FIG. 2 shows a Sample Dose Response Curve of the Positive ControlChlorpromazine for use in PIF calculations.

FIG. 3 shows a Sample Dose Response Curve for use in MPE calculations.

DETAILED DESCRIPTION

The present invention provides a method for simultaneously treatingocular rosacea and acne rosacea.

As used herein, the term “ocular rosacea” is a disorder characterized byeye surface inflammation, and a variety of related eye disorders such asblepharitis; meibomian gland disease, including meibomianitis;keratitis; conjunctival hyperemia; and eyelid hyperemia. The eye surfaceincludes the eyelids, cornea and conjunctiva.

The present invention is particularly effective in treating all knowntypes of blepharitis. Some types of blepharitis include, for example,blepharitis angularis, blepharitis ciliaris, blepharitis marginalis,nonulcerative blepharitis, seborrheic blepharitis, blepharitis squamosa,squamous seborrheic blepharitis, and blepharitis ulcerosa.

Acne rosacea is a skin condition characterized by inflammatory lesions(erythema) and permanent dilation of blood vessels (telangectasia). Acnerosacea can also include papules, pustules, and hypertrophic sebaceousglands in facial flush areas. A manifestation of severe acne rosacea isrhinophyma. Rhinophyma is characterized by a thickened, lobulatedovergrowth of the sebaceous glands and epithelial connective tissue ofthe nose.

The method of the present invention comprises the administration of atetracycline compound to a human in an amount which is effective for itspurpose e.g., the simultaneous treatment of ocular rosacea and acnerosacea, but which has substantially no antibiotic activity. Preferably,the human is monitored. Monitoring is accomplished by observing apositive result. A positive result includes reducing or reversing thesymptoms characterizing ocular rosacea and acne rosacea.

The tetracycline compound can be an antibiotic or non-antibioticcompound. The tetracyclines are a class of compounds of whichtetracycline is the parent compound. Tetracycline has the followinggeneral structure:

The numbering system of the multiple ring nucleus is as follows:

Tetracycline, as well as the 5-hydroxy(oxytetracycline, e.g. Terramycin)and 7-chloro(chlorotetracycline, e.g. Aureomycin) derivatives, exist innature, and are all well known antibiotics. Semisynthetic derivativessuch as 7-dimethylaminotetracycline (minocycline) and6α-deoxy-5-hydroxytetracycline (doxycycline) are also known tetracyclineantibiotics. Natural tetracyclines may be modified without losing theirantibiotic properties, although certain elements of the structure mustbe retained to do so.

Some examples of antibiotic (i.e. antimicrobial) tetracycline compoundsinclude doxycycline, minocycline, tetracycline, oxytetracycline,chlortetracycline, demeclocycline, lymecycline and theirpharmaceutically acceptable salts. Doxycycline is preferablyadministered as its hyclate salt or as a hydrate, preferablymonohydrate.

Non-antibiotic tetracycline compounds are structurally related to theantibiotic tetracyclines, but have had their antibiotic activitysubstantially or completely eliminated by chemical modification. Forexample, non-antibiotic tetracycline compounds are capable of achievingantibiotic activity comparable to that of tetracycline or doxycycline atconcentrations at least about ten times, preferably at least abouttwenty five times, greater than that of tetracycline or doxycycline,respectively.

Examples of chemically modified non-antibiotic tetracyclines (CMTs)include 4-de(dimethylamino)tetracycline (CMT-1), tetracyclinonitrile(CMT-2), 6-demethyl-6-deoxy-4-de(dimethylamino)tetracycline (CMT-3),7-chloro-4-de(dimethylamino)tetracycline (CMT-4), tetracycline pyrazole(CMT-5), 4-hydroxy-4-de(dimethylamino)tetracycline (CMT-6),4-de(dimethylamino-12α-deoxytetracycline (CMT-7),6-deoxy-5α-hydroxy-4-de(dimethylamino)tetracycline (CMT-8),4-de(dimethylamino)-12α-deoxyanhydrotetracycline (CMT-9),4-de(dimethylamino)minocycline (CMT-10). (COL and CMT are usedinterchangeably throughout this specification.)

Further examples of chemically modified non-antibiotic tetracyclinesinclude Structures C—Z. (See Index of Structures.)

Tetracycline derivatives, for purposes of the invention, may be anytetracycline derivative, including those compounds disclosed genericallyor specifically in co-pending U.S. patent application Ser. No.09/573,654, filed on May 18, 2000; and Ser. No. 10/274,841, filed onOct. 18, 2002, which are incorporated herein by reference.

The tetracycline compound is administered in an amount which iseffective to simultaneously treat ocular rosacea and acne rosacea, butwhich has substantially no antibiotic effect. A treatment is effectiveif it causes a reduction or inhibition of the symptoms associated withocular rosacea and acne rosacea.

The minimal effective amount of a tetracycline compound administered toa human is the lowest amount capable of providing effective simultaneoustreatment of ocular rosacea and acne rosacea. Some examples of minimalamounts include 10%, 20%, 30% and 40% of an antibiotic amount.

The maximal effective amount of a tetracycline compound administered toa human is the highest amount that does not significantly prevent thegrowth of microbes, e.g., bacteria. Some examples of maximal amountsinclude 50%, 60%, 70% and 80% of an antibiotic amount.

The amount of a tetracycline compound which is administered can bemeasured by daily dose and by serum level.

Tetracycline compounds that have significant antibiotic activity may,for example, be administered in a dose which is 10-80% of the antibioticdose. More preferably, the antibiotic tetracycline compound isadministered in a dose which is 40-70% of the antibiotic dose.

Antibiotic daily doses are known in art. Some examples of antibioticdoses of members of the tetracycline family include 50, 75, and 100mg/day of doxycycline; 50, 75, 100, and 200 mg/day of minocycline; 250mg of tetracycline one, two, three, or four times a day; 1000 mg/day ofoxytetracycline; 600 mg/day of demeclocycline; and 600 mg/day oflymecycline.

Examples of the maximum non-antibiotic doses of tetracyclines based onsteady-state pharmacokinetics are as follows: 20 mg/twice a day fordoxycycline; 38 mg of minocycline one, two, three or four times a day;and 60 mg of tetracycline one, two, three or four times a day.

In a preferred embodiment, doxycycline is administered in a daily amountof from about 30 to about 60 milligrams, but maintains a concentrationin human plasma below the threshold for a significant antibiotic effect.

In an especially preferred embodiment, doxycycline hyclate isadministered at a 20 milligram dose twice daily. Such a formulation issold for the treatment of periodontal disease by CollaGenexPharmaceuticals, Inc. of Newtown, Pa. under the trademark Periostat®.

The administered amount of a tetracycline compound described by serumlevels follows.

An antibiotic tetracycline compound is advantageously administered in anamount that results in a serum tetracycline concentration which is10-80%, preferably 40-70%, of the minimum antibiotic serumconcentration. The minimum antibiotic serum concentration is the lowestconcentration known to exert a significant antibiotic effect.

Some examples of the approximate antibiotic serum concentrations ofmembers of the tetracycline family follow. A single dose of two 100 mgminocycline HCl tablets administered to adult humans results inminocycline serum levels ranging from 0.74 to 4.45 μg/ml over a periodof an hour. The average level is 2.24 μg/ml.

Two hundred and fifty milligrams of tetracycline HCl administered everysix hours over a twenty-four hour period produces a peak plasmaconcentration of approximately 3 μg/ml. Five hundred milligrams oftetracycline HCl administered every six hours over a twenty-four hourperiod produces a serum concentration level of 4 to 5 μg/ml.

In one embodiment, the tetracycline compound can be administered in anamount which results in a serum concentration between about 0.1 and 10.0μg/ml, more preferably between 0.3 and 5.0 μg/ml. For example,doxycycline is administered in an amount which results in a serumconcentration between about 0.1 and 0.8 μg/ml, more preferably between0.4 and 0.7 μg/ml.

Some examples of the plasma antibiotic threshold levels of tetracyclinesbased on steady-state pharmacokinetics are as follows: 1.0 μg/ml fordoxycycline; 0.8 μg/ml for minocycline; and 0.5 μg/ml for tetracycline.

Non-antibiotic tetracycline compounds can be used in higher amounts thanantibiotic tetracyclines, while avoiding the indiscriminate killing ofmicrobes, and the emergence of resistant microbes. For example,6-demethyl-6-deoxy-4-de(dimethylamino)tetracycline (CMT-3) may beadministered in doses of about 40 to about 200 mg/day, or in amountsthat result in serum levels of about 1.55 μg/ml to about 10 μg/ml.

The actual preferred amounts of tetracycline compounds in a specifiedcase will vary according to the particular compositions formulated, themode of application, the particular sites of application, and thesubject being treated (e.g. age, gender, size, tolerance to drug, etc.)

The tetracycline compounds can be in the form of pharmaceuticallyacceptable salts of the compounds. The term “pharmaceutically acceptablesalt” refers to a salt prepared from tetracycline compounds andpharmaceutically acceptable non-toxic acids or bases. The acids may beinorganic or organic acids of tetracycline compounds. Examples ofinorganic acids include hydrochloric, hydrobromic, nitric hydroiodic,sulfuric, and phosphoric acids. Examples of organic acids includecarboxylic and sulfonic acids. The radical of the organic acids may bealiphatic or aromatic. Some examples of organic acids include formic,acetic, phenylacetic, propionic, succinic, glycolic, glucuronic, maleic,furoic, glutamic, benzoic, anthranilic, salicylic, phenylacetic,mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic, panthenoic,benzenesulfonic, stearic, sulfanilic, alginic, tartaric, citric,gluconic, gulonic, arylsulfonic, and galacturonic acids. Appropriateorganic bases may be selected, for example, fromN,N-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,ethylenediamine, meglumine (N-methylglucamine), and procaine.

The tetracycline compounds mentioned above, especially doxycycline andminocycline, are unexpectedly effective in reducing the number ofcomedones when administered at a dose which has substantially noantibiotic effect. Preferably the reduction is at least about 20%greater than for a placebo control, more preferably at least about 30%greater than for a placebo control, most preferably at least about 40%greater than for a placebo control, and optimally at least about 50%greater than for a placebo control.

Preferably, the tetracycline compounds have low phototoxicity, or areadministered in an amount that results in a serum level at which thephototoxicity is acceptable. Phototoxicity is a chemically-inducedphotosensitivity. Such photosensitivity renders skin susceptible todamage, e.g. sunburn, blisters, accelerated aging, erythemas andeczematoid lesions, upon exposure to light, in particular ultravioletlight. The preferred amount of the tetracycline compound produces nomore phototoxicity than is produced by the administration of a 40 mgtotal daily dose of doxycycline.

There are several methods by which to quantify phototoxicity. One methodis called photoirritancy factor (PIF). The PIF is the ratio of an IC₅₀value in the absence of light to an IC₅₀ value in the presence of light.

In calculating PIF values, the data resulting from the assay procedurecan be interpreted by different methods. For example, during the periodMar. 2, 1999 to Apr. 16, 1999, PIF values were obtained using thephototoxicity software and its curve-fitting algorithms available at thetime. In the present specification, this earlier phototoxicitycalculation is referred to as PIF1. At a PIF1 value of 1, a compound isconsidered to have no measurable phototoxicity. A PIF1 value greaterthan 5 is indicative of phototoxic potential of a compound.

As explained in more detail in Example 37 below, 3T3 phototoxicity assayhas undergone extensive validation since April 1999, and has now beenincorporated into a draft guideline by the Organization of EconomicCooperation and Development (OECD) (Draft Guideline 432). In the presentspecification, this revised phototoxicity calculation is referred to asPIF2. A PIF2 value of less than 2 is considered non-phototoxic, 2 toless than 5 is considered potentially phototoxic, and 5 or greater isconsidered clearly phototoxic.

PIF2 values are more refined than the PIF1 values. Qualitatively thedifferences between the PIF1 and PIF2 values are not significant. Forexample, the mean PIF1 values for COL 10 and COL 1002 are 1.82 and 1.0,respectively. The mean PIF2 values of COL 10 and COL 1002 are 2.04 and1.35, respectively.

As explained in the Examples section, PIF values cannot be determinedfor many compounds. Another method by which to quantify relativephototoxicity is called mean photo effect (MPE). MPE values can bedetermined for compounds in virtually all cases. Thus, MPE values aremore consistent and reliable than PFE values.

The MPE is a measure of the difference between the cytotoxicity inducedby the test chemical in the presence and absence of light. It comparesthe responses over the range of doses selected using the twodose-response curves produced from the boot-strap analysis of theindividual data points (Holzhütter 1995 and 1997). An example isprovided in FIG. 3 (Peters and Holzhütter (2002)). This method ofanalysis is particularly suited to cases where the IC₅₀ value cannot becalculated for one or both concentration response curves.

MPE values of <0.1 (including negative values) are considered indicativeof a nonphototoxin, values of 0.1 to <0.15 are considered probablephototoxins, and values greater than and equal to 0.15 are considered tobe clear phototoxins.

A class of low phototoxicity tetracyline derivatives has less thanapproximately 75% of the phototoxicity of minocycline, preferably lessthan approximately 70%, more preferably less than approximately 60%, andmost preferably less than approximately 50%. Minocycline has a PIF1 ofabout 2.04, and an MPE of about 0.041.

The class of low phototoxicity tetracycline compound derivativesincludes those derivatives having PIF1 or PIF 2 values of approximately1, i.e. 1 to about 2, preferably 1 to about 1.5. The class of lowphototoxicity tetracycline derivatives optimally have MPE values of lessthan 0.1. Members of this class include, but are not limited to,tetracycline compounds having general formulae:

Structure K

wherein: R7, R8, and R9 taken together in each case, have the followingmeanings:

R7 R8 R9 hydrogen hydrogen amino hydrogen hydrogen palmitamide hydrogenhydrogen dimethylamino trimethylammonium hydrogen hydrogenand

STRUCTURE L STRUCTURE M STRUCTURE N STRUCTURE O

wherein: R7, R8, and R9 taken together in each case, have the followingmeanings:

R7 R8 R9 hydrogen hydrogen acetamido hydrogen hydrogendimethylaminoacetamido hydrogen hydrogen nitro hydrogen hydrogen aminoand

Structure P

wherein: R8, and R9 taken together are, respectively, hydrogen andnitro.

The tetracycline compounds may, for example, be administeredsystemically. For the purposes of this specification, “systemicadministration” means administration to a human by a method that causesthe compounds to be absorbed into the bloodstream.

For example, the tetracyclines compounds can be administered orally byany method known in the art. For example, oral administration can be bytablets, capsules, pills, troches, elixirs, suspensions, syrups, wafers,chewing gum and the like.

Additionally, the tetracycline compounds can be administered enterallyor parenterally, e.g., intravenously; intramuscularly; subcutaneously,as injectable solutions or suspensions; intraperitoneally; or rectally.Administration can also be intranasally, in the form of, for example, anintranasal spray; or transdermally, in the form of, for example, apatch.

For the pharmaceutical purposes described above, the tetracyclinecompounds of the invention can be formulated per se in pharmaceuticalpreparations optionally with a suitable pharmaceutical carrier (vehicle)or excipient as understood by practitioners in the art. Thesepreparations can be made according to conventional chemical methods.

In the case of tablets for oral use, carriers which are commonly usedinclude lactose and corn starch, and lubricating agents such asmagnesium stearate are commonly added. For oral administration incapsule form, useful carriers include lactose and corn starch. Furtherexamples of carriers and excipients include milk, sugar, certain typesof clay, gelatin, stearic acid or salts thereof, calcium stearate, talc,vegetable fats or oils, gums and glycols.

When aqueous suspensions are used for oral administration, emulsifyingand/or suspending agents are commonly added. In addition, sweeteningand/or flavoring agents may be added to the oral compositions.

For intramuscular, intraperitoneal, subcutaneous and intravenous use,sterile solutions of the tetracycline compounds can be employed, and thepH of the solutions can be suitably adjusted and buffered. Forintravenous use, the total concentration of the solute(s) can becontrolled in order to render the preparation isotonic.

The tetracycline compounds of the present invention can further compriseone or more pharmaceutically acceptable additional ingredient(s) such asalum, stabilizers, buffers, coloring agents, flavoring agents, and thelike.

The tetracycline compound may be administered intermittently. Forexample, the tetracycline compound may be administered 1-6 times a day,preferably 1-4 times a day.

Alternatively, the tetracycline compound may be administered bysustained release. Sustained release administration is a method of drugdelivery to achieve a certain level of the drug over a particular periodof time. The level typically is measured by serum concentration. Furtherdescription of methods of delivering tetracycline compounds by sustainedrelease can be found in the patent application, “Controlled Delivery ofTetracycline and Tetracycline Derivatives,” filed on Apr. 5, 2001 andassigned to CollaGenex Pharmaceuticals, Inc. of Newtown, Pa. Theaforementioned application is incorporated herein by reference in itsentirety. For example, 40 milligrams of doxycycline may be administeredby sustained release over a 24 hour period.

In the embodiment in which the tetracycline compound is a non-antibiotictetracycline compound, administration can include topical application tothe skin and eye. Particular non-antibiotic tetracycline compounds haveonly limited biodistribution, e.g. CMT-5. In such cases, topicalapplication is the preferred method of administration of the compound.

Carrier compositions deemed to be suited for topical use include gels,salves, lotions, creams, ointments, eye drops and the like. Thenon-antibiotic tetracycline compound can also be incorporated with asupport base or matrix or the like which can be directly applied to skinor the eye. The carrier compositions used to topically treat acnerosacea and ocular rosacea can be the same, or can be different. Forexample, the carrier composition used to simultaneously treat acnerosacea and ocular rosacea can both be gels. Alternatively, for example,the carrier composition used to treat acne rosacea can be an ointment,while the carrier composition used to treat ocular rosacea can be in eyedrop form.

Topical application of the non-antibiotic tetracycline compounds areeffective in simultaneously treating acne rosacea and ocular rosaceawhile not inducing significant toxicity in the human. For example,amounts of up to about 25% (w/w) in a vehicle are effective. Amounts offrom about 0.1% to about 10% are preferred.

Combined or coordinated topical and systemic administration of thetetracycline compounds is also contemplated under the invention. Forexample, a non-absorbable non-antibiotic tetracycline compound can beadministered topically, while a tetracycline compound capable ofsubstantial absorption and effective systemic distribution in a humancan be administered systemically.

The tetracycline compounds are prepared by methods known in the art. Forexample, natural tetracyclines may be modified without losing theirantibiotic properties, although certain elements of the structure mustbe retained. The modifications that may and may not be made to the basictetracycline structure have been reviewed by Mitscher in The Chemistryof Tetracyclines, Chapter 6, Marcel Dekker, Publishers, New York (1978).According to Mitscher, the substituents at positions 5-9 of thetetracycline ring system may be modified without the complete loss ofantibiotic properties. Changes to the basic ring system or replacementof the substituents at positions 1-4 and 10-12, however, generally leadto synthetic tetracyclines with substantially less or effectively noantibiotic activity.

Further methods of preparing the tetracycline compounds are described inthe examples.

EXAMPLES

The following examples serve to provide further appreciation of theinvention but are not meant in any way to restrict the effective scopeof the invention.

Preparation of Compounds Example 14-Dedimethylamino-7-dimethylamino-6-demethyl-6-deoxy-9-nitrotetracyclinesulfate

To a solution of one millimole of4-dedimethylamino-7-dimethylamino-6-demethyl-6-deoxytetracycline in 25ml of concentrated sulfuric acid at 0° C. was added 1.05 mmole ofpotassium nitrate. The resulting solution was stirred at ice bathtemperature for 15 minutes and poured in one liter of cold ether withstirring. The precipitated solid was allowed to settle and the majorityof solvent decanted. The remaining material was filtered through asintered glass funnel and the collected solid was washed well with coldether. The product was dried in a vacuum desiccator overnight.

Example 29-amino-4-dedimethylamino-7-dimethylamino-6-demethyl-6-deoxytetracyclinesulfate

To a solution of 300 mg of the 9-nitro compound from example 1, in 30 mlof ethanol was added 50 mg of PtO₂. The mixture was hydrogenated atatmospheric pressure until the theoretical amount of hydrogen wasabsorbed. The system is flushed with nitrogen, the catalyst PtO₂ isfiltered and the filtrate added dropwise to 300 ml of ether. The productthat separates is filtered and dried in a vacuum desiccator.

Example 39-Acetamido-4-dedimethylamino-7-dimethylamino-6-demethyl-6-deoxytetracyclinesulfate

To a well stirred cold solution of 500 mg of9-amino-4-dedimethylamino-7-dimethylamino-6-demethyl-6-deoxytetracyclinesulfate from example 2, in 2.0 ml of 1.3-dimethyl-2-imidazolidinone, 500mg of sodium bicarbonate was added followed by 0.21 ml of acetylchloride. The mixture is stirred at room temperature for 30 minutes,filtered and the filtrate was added dropwise to 500 ml of ether. Theproduct that separated was filtered and dried in a vacuum desiccator.

Example 44-Dedimethylamino-7-dimethylamino-6-demethyl-6-deoxy-9-diazoniumtetracyclinesulfate

To a solution of 0.5 g of9-amino-4-dedimethylamino-7-dimethylamino-6-demethyl-6-deoxytetracyclinesulfate, from example 2, in 10 ml of 0.1N hydrochloric acid in methanolcooled in an ice bath, 0.5 ml of n-butyl nitrite was added. The solutionwas stirred at ice bath temperature for 30 minutes and then poured into250 ml of ether. The product that separated was filtered, washed withether and dried in a vacuum desiccator.

Example 59-Azido-4-dedimethylamino-7-dimethylamino-6-demethyl-6-deoxytetracyclinesulfate

To a solution of 0.3 mmole of4-dedimethylamino-7-dimethylamino-6-demethyl-6-deoxy-9-diazoniumtetracyclinesulfate, from example 4, 10 ml of 0.1N methanolic hydrogen chloride wasadded 0.33 mmole of sodium azide. The mixture was stirred at roomtemperature for 1.5 hours. The reaction mixture was then poured into 200ml of ether. The product that separated was filtered and dried in avacuum desiccator.

Example 69-Amino-8-chloro-4-dedimethylamino-7-dimethylamino-6-demethyl-6-deoxy-tetracyclinesulfate

One gram of9-azido-4-dedimethylamino-7-dimethylamino-6-demethyl-6-deoxytetracyclinehydrochloride, from example 4, was dissolved in 10 ml of concentratedsulfuric acid saturated with HCL at 0° C. The mixture was stirred at icebath temperature for 1.5 hours and then slowly added dropwise to 500 mlof cold ether. The product that separated was filtered, washed withether and dried in a vacuum desiccator.

Example 74-Dedimethylamino-7-dimethylamino-6-demethyl-6-deoxy-9-ethoxythiocarbonylthio-tetracyclinesulfate

A solution of 1.0 mmole of4-dedimethylamino-7-dimethylamino-6-demethyl-6-deoxy-9-diazoniumtetracyclinesulfate, from example 4, in 15 ml of water was added to a solution of1.15 mmole of potassium ethyl xanthate in 15 ml of water. The mixturewas stirred at room temperature for one hour. The product separated andwas filtered and dried in a vacuum desiccator.

Example 8A General Procedure for Nitration

To 1 mmole of a 4-dedimethylamino-6-deoxytetracycline in 25 ml ofconcentrated sulfuric acid at 0° C. was added 1 mmole of potassiumnitrate with stirring. The reaction solution was stirred for 15 minutesand then poured into 100 g of chopped ice. The aqueous solution wasextracted 5 times with 20 ml of butanol each time. The butanol extractswere washed three times with 10 ml of water each time, and concentratedin vacuo to a volume of 25 ml. The light yellow crystalline solid whichprecipitated was filtered, washed with 2 ml of butanol and dried invacuo at 60° C. for 2 hours. This solid was a mixture of the twomononitro isomers.

Example 8B 4-Dedimethylamino-6-deoxy-9-nitrotetracycline

To 980 mg of the nitration product from4-dedimethylamino-6-deoxytetracycline (a mixture of the 2 isomers) in 25ml of methanol was added enough triethylamine to dissolve the solid. Thefiltered solution (pH 9.0) was adjusted to pH 5.2 with concentratedsulfuric acid. A crystalline yellow solid (236 mg.) was obtained (29%yield). The material at this point was quite pure and contained onlysmall amounts of the 7-isomer. Final purification was accomplished byliquid partition chromatography using a diatomaceous earth packed columnand the solvent system: chloroform: butanol: 0.5M phosphate buffer (pH2) (16:1:10).

Example 9 4-Dedimethylamino-6-deoxy-7-nitrotetracycline

The methanol filtrate from example 8 was immediately adjusted to pH 1.0with concentrated sulfuric acid. The light yellow crystalline solid,which was obtained as the sulfate salt. A purified free base wasobtained by adjusting an aqueous solution of the sulfate salt (25 mg/ml)to pH 5.2 with 2 N sodium carbonate.

Example 10 9-Amino-4-dedimethylamino-6-deoxytetracycline

To a solution of 300 mg of the 9-nitro compound, prepared in example 8,in 30 ml of ethanol was added 50 mg of PtO₂. The mixture washydrogenated at atmospheric pressure until the theoretical amount ofhydrogen was absorbed. The system is flushed with nitrogen, the PtO₂catalyst is filtered and the filtrate added dropwise to 300 ml of ether.The solid that separates is filtered and dried in a vacuum desiccator.

Example 11 9-Acetamido-4-dedimethylamino-6-deoxytetracycline sulfate

To well stirred cold solution of 500 mg of9-amino-4-dedimethylamino-6-deoxytetracycline sulfate, from example 10,in 2.0 ml of 1,3-dimethyl-2-imidazolidinone was added 500 mg of sodiumbicarbonate followed by 0.21 ml of acetyl chloride. The mixture wasstirred at room temperature for 30 minutes, filtered and the filtratewas added dropwise to 500 ml of ether. The solid that separated wasfiltered and dried in a vacuum desiccator.

Example 12 4-Dedimethylamino-6-deoxy-9-diazoniumtetracycline sulfate

To a solution of 0.5 g of 9-amino-4-dedimethylamino-6-deoxytetracyclinesulfate, from example 10, in 10 ml of 0.1N hydrochloric acid in methanolcooled in an ice bath was added 0.5 ml of n-butyl nitrite. The solutionwas stirred at ice bath temperature for 30 minutes and the poured into250 ml of ether. The solid that separated was filtered, washed withether and dried in a vacuum desiccator.

Example 13 9-Azido-4-dedimethylamino-6-deoxytetracycline sulfate

To a solution of 0.3 mmole of4-dedimethylamino-6-deoxy-9-diazoniumtetracycline sulfate, of example12, 10 ml of 0.1N methanolic hydrogen chloride was added 0.33 mmole ofsodium azide. The mixture was stirred at room temperature for 1.5 hours.The reaction mixture was then poured into 200 ml of ether. The solidthat separated was filtered and dried in a vacuum desiccator.

Example 14 9-Amino-8-chloro-4-dedimethylamino-6-deoxytetracyclinesulfate

One gram of9-azido-4-dedimethylamino-7-dimethylamino-6-deoxytetracyclinehydrochloride, from example 13, was dissolved in 10 ml of concentratedsulfuric acid saturated with HCL at 0° C. The mixture was stirred at icebath temperature for 1.5 hours and then slowly added dropwise to 500 mlof cold ether. The solid that separated was filtered, washed and etherand dried in a vacuum desiccator.

Example 154-Dedimethylamino-6-deoxy-9-ethoxythiocarbonylthiotetracycline sulfate

A solution of 1.0 mmole of4-dedimethylamino-6-deoxy-9-diazoniumtetracycline sulfate, from example12, in 15 ml of water was added to a solution of 1.15 mmole of potassiumethyl xanthate in 15 ml of water. The mixture was stirred at roomtemperature for one hour. The solid that separated was filtered anddried in a vacuum desiccator.

Example 16 9-Dimethylamino-4-dedimethylamino-6-deoxytetracycline sulfate

To a solution of 100 mg. of the 9-amino compound from example 10, in 10ml of ethylene glycol monomethyl ether is added 0.05 ml of concentratedsulfuric acid, 0.4 ml. of a 40% aqueous formaldehyde solution and 100 mgof a 10% palladium on carbon catalyst. The mixture is hydrogenated underatmospheric pressure and room temperature for 20 minutes. The catalystwas filtered and the filtrate was evaporated to dryness under reducedpressure. The residue is dissolved in 5 ml of methanol and this solutionwas added to 100 ml of ether. The product that separated was filteredand dried, yield, 98 mg.

Example 17 7-Amino-4-dedimethylamino-6-deoxytetracycline

This compound can be made using Procedure A or B. Procedure A. To asolution of 300 mg of the 7-nitro compound, from example 1, in 30 ml ofethanol was added 50 mg of PtO₂. The mixture was hydrogenated atatmospheric pressure until the theoretical amount of hydrogen wasabsorbed. The system is flushed with nitrogen, the catalyst PtO₂ isfiltered and the filtrate added dropwise to 300 ml of ether. The solidthat separates is filtered and dried in a vacuum desiccator.

Procedure B. 1 g of 6-deoxy-4-dedimethylamino-tetracycline was dissolvedin 7.6 ml THF and 10.4 ml methanesulfonic acid at −10° C. After warmingthe mixture to 0° C. a solution of 0.86 g of dibenzyl azodicarboxylatewas added and the mixture stirred for 2 hours at 0° C. to yield7-[1,2-bis(carbobenzyloxy)hydrazino]-4-dedimethylamino-6-deoxytetracycline.A solution of 1 millimole of this material in 70 ml 2-methoxyethanol,and 300 mg 10% Pd—C was hydrogenated at room temperature to give7-amino-6-deoxy-4-dedimethylaminotetracycline.

Example 18 7-Amino-6-deoxy-5-hydroxy-4-dedimethylaminotetracycline

1 g of 6-deoxy-5-hydroxy-4-dedimethylaminotetracycline 3 was dissolvedin 7.6 ml THF and 10.4 ml methanesulfonic acid at −10° C. After warmingthe mixture to 0° C. a solution of 0.86 g dibenzyl azodicarboxylate in0.5 ml THF was added and the mixture stirred for 2 hours at 0° C. toyield7-[1,2-bis(carbobenzyloxy)hydrazino]-4-dedimethylamino-6-deoxy-5-hydroxytetracycline.A solution of 1 millimole of this material in 70 ml 2-methoxyethanol,and 300 mg 10% Pd—C was hydrogenated at room temperature to give7-amino-6-deoxy-5-hydroxytetracycline.

Example 19 7-Acetamido-4-dedimethylamino-6-deoxy-5-hydroxytetracyclinesulfate

To well stirred cold solution of 500 mg of7-amino-4-dedimethylamino-6-deoxy-5-hydroxytetracycline sulfate, fromexample 18, in 2.0 ml of 1,3-dimethyl-2-imidazolidinone was added 500 mgof sodium bicarbonate followed by 0.21 ml of acetyl chloride. Themixture was stirred at room temperature for 30 minutes, filtered and thefiltrate was added dropwise to 500 ml of ether. The solid that separatedwas filtered and dried in a vacuum desiccator.

Example 20 4-Dedimethylamino-6-deoxy-5-hydroxy-7-diazoniumtetracyclinehydrochloride

To a solution of 0.5 g of7-amino-4-dedimethylamino-6-deoxy-5-hydroxytetracycline sulfate, fromexample 20, in 10 ml of 0.1N hydrochloric acid in methanol cooled in anice bath was added 0.5 ml of n-butyl nitrite. The solution was stirredat ice bath temperature for 30 minutes and then poured into 250 ml ofether. The solid that separated was filtered, washed with ether anddried in a vacuum desiccator.

Example 21 7-Azido-4-dedimethylamino-6-deoxy-5-hydroxytetracycline

To a solution of 0.3 mmole of4-dedimethylamino-6-deoxy-5-hydroxy-7-diazoniumtetracyclinehydrochloride, from example 20, 10 ml of 0.1N methanolic hydrogenchloride was added 0.33 mmole of sodium azide. The mixture was stirredat room temperature for 1.5 hours. The reaction mixture was then pouredinto 200 ml of ether. The solid that separated was filtered and dried ina vacuum desiccator.

Example 227-Amino-8-chloro-4-dedimethylamino-6-deoxy-5-hydroxytetracycline sulfate

One gram of7-azido-4-dedimethylamino-7-dimethylamino-6-deoxy-5-hydroxytetracyclinesulfate, from example 21, was dissolved in 10 ml of concentratedsulfuric acid (previously saturated with hydrogen chloride) at 0° C. Themixture was stirred at ice bath temperature for 1.5 hours and thenslowly added dropwise to 500 ml of cold ether. The solid that separatedwas filtered, washed with ether and dried in a vacuum desiccator.

Example 234-Dedimethylamino-6-deoxy-5-hydroxy-7-ethoxythiocarbonylthiotetracycline

A solution of 1.0 mmole of4-dedimethylamino-6-deoxy-5-hydroxy-7-diazoniumtetracyclinehydrochloride, from example 20, in 15 ml of water was added to asolution of 1.15 mmole of potassium ethyl xanthate in 15 ml of water.The mixture was stirred at room temperature for one hour. The solid thatseparated was filtered and dried in a vacuum desiccator.

Example 247-Dimethylamino-4-dedimethylamino-6-deoxy-5-hydroxytetracycline sulfate

To a solution of 100 mg of the 7-amino compound in 10 ml of ethyleneglycol monomethyl ether is added 0.05 ml of concentrated sulfuric acid,0.4 ml of a 40% aqueous formaldehyde solution and 100 mg of a 10%palladium on carbon catalyst. The mixture is reduced with hydrogen atatmospheric pressure and room temperature for 20 minutes. The catalystwas filtered and the filtrate was evaporated to dryness under reducedpressure. The residue is dissolved in 5 ml of methanol and this solutionwas added to 100 ml of ether. The product that separated was filteredand dried, yield, 78 mg.

Example 25 7-Diethylamino-4-dedimethylamino-5-hydroxytetracyclinesulfate

To a solution of 100 mg of the 7-amino compound in 10 ml of ethyleneglycol monomethyl ether is added 0.05 ml of concentrated sulfuric acid,0.4 ml of acetaldehyde and 100 mg of a 10% palladium on carbon catalyst.The mixture is reduced with hydrogen at atmospheric pressure at roomtemperature for 20 minutes. The catalyst was filtered and filtrate wasevaporated to dryness under reduced pressure. The residue is dissolvedin 5 ml of methanol and this solution was added to 100 ml of ether. Theproduct that separated was filtered and dried.

Example 26 4-Dedimethylamino-6-deoxy-7-diazoniumtetracyclinehydrochloride

To a solution of 0.5 g. of 7-amino-4-dedimethylamino-6-deoxytetracyclinesulfate, from example 17, in 10 ml of 0.1N hydrochloric acid in methanolcooled in an ice bath was added 0.5 ml of n-butyl nitrite. The solutionwas stirred at ice bath temperature for 30 minutes and then poured into250 ml of ether. The solid that separated was filtered, washed withether and dried in a vacuum desiccator.

Example 27 7-Azido-4-dedimethylamino-6-deoxytetracycline

To a solution of 0.3 mmole of4-dedimethylamino-6-deoxy-7-diazoniumtetracycline hydrochloride, fromexample 26, 10 ml of 0.1N methanolic hydrogen chloride was added 0.33mmole of sodium azide. The mixture was stirred at room temperature for1.5 hours. The reaction mixture was then poured into 200 ml of ether.The solid that separated was filtered and dried in a vacuum desiccator.

Example 28 7-Amino-8-chloro-4-dedimethylamino-6-deoxytetracyclinesulfate

One gram of7-azido-4-dedimethylamino-7-dimethylamino-6-deoxytetracycline sulfatewas dissolved in 10 ml of concentrated sulfuric acid (previouslysaturated with hydrogen chloride) at 0° C. The mixture was stirred atice bath temperature for 1.5 hours and then slowly added dropwise to 500ml of cold ether. The solid that separated was filtered, washed withether and dried in a vacuum desiccator.

Example 294-Dedimethylamino-6-deoxy-7-ethoxythiocarbonylthiotetracycline

A solution of 1.0 mmole of4-dedimethylamino-6-deoxy-7-diazoniumtetracycline hydrochloride, fromexample 26, in 15 ml of water was added to a solution of 1.15 mmole ofpotassium ethyl xanthate in 15 ml of water. The mixture was stirred atroom temperature for one hour. The solid that separated was filtered anddried in a vacuum desiccator.

Example 30 7-Dimethylamino-4-dedimethylamino-6-deoxytetracycline sulfate

To a solution of 100 mg of the 7-amino compound, from example 26, in 10ml of ethylene glycol monomethyl ether is added 0.05 ml of concentratedsulfuric acid, 0.4 ml of a 40% aqueous formaldehyde solution and 100 mgof a 10% palladium on carbon catalyst. The mixture is reduced withhydrogen at atmospheric pressure and room temperature for 20 minutes.The catalyst was filtered and the filtrate was evaporated to drynessunder reduced pressure. The residue is dissolved in 5 ml of methanol andthis solution was added to 100 ml of ether. The product that separatedwas filtered and dried.

Example 319-Acetamido-8-chloro-4-dedimethylamino-7-dimethylamino-6-deoxy-6-demethyltetracycline

To well stirred cold solution of 500 mg of9-amino-8-chloro-4-dedimethylamino-6-deoxy-6-demethyl-7-dimethyl aminotetracycline sulfate, from example 6, in 2.0 ml of1,3-dimethyl-2-imidazolidinone was added 500 mg of sodium bicarbonatefollowed by 0.21 ml. of acetyl chloride. The mixture was stirred at roomtemperature for 30 minutes, filtered and the filtrate was added dropwiseto 500 ml of ether. The solid that separated was filtered and dried in avacuum desiccator.

Example 328-Chloro-4-dedimethylamino-7-dimethylamino-6-deoxy-6-demethyl-9-ethoxythiocarbonylthiotetracycline

A solution of 1.0 mmole of−8-chloro-4-dedimethylamino-6-deoxy-6-demethyl-7-dimethylamino-9-diazoniumtetracycline hydrochloride in 15 ml of water was addedto a solution of 1.15 mmole of potassium ethyl xanthate in 15 ml ofwater. The mixture was stirred at room temperature for one hour. Thesolid that separated was filtered and dried in a vacuum desiccator.

Example 338-Chloro-9-dimethylamino-4-dedimethylamino-7-dimethylamino-6-deoxy-6-demethyletracyclinesulfate

To a solution of 100 mg. of the 9-amino compound, from example 6, in 10ml of ethylene glycol monomethyl ether is added 0.05 ml of concentratedsulfuric acid, 0.4 ml of acetaldehyde and 100 mg of a 10% palladium oncarbon catalyst. The mixture is reduced with hydrogen at atmosphericpressure and room temperature for 20 minutes. The catalyst was filteredand the filtrate was evaporated to dryness under reduced pressure. Theresidue is dissolved in 5 ml of methanol and this solution was added to100 ml of ether. The product that separated was filtered and dried.

Example 34 N-(4-methylpiperazin-1-yl)methyl-4-dedimethylamino-6-demethyl-6-deoxytetracycline

An aqueous solution of 58 mg (37%) formaldehyde (0.72 mmol) was added toa solution of 203 mg (0.49 mmol) of4-dedimethylamino-6-demethyl-6-deoxytetracycline in 5.0 ml ethyleneglycol dimethyl ether. The mixture was stirred at room temperature for0.5 hours. 56 mg (0.56 mmol) of 1-methylpiperazine was then added andthe resulting mixture was stirred overnight and refluxed for 20 minutes.The mixture was then cooled and a solid product was collected byfiltration. The solid product was then washed with the solvent and driedby vacuum filtration.

Example 35N-(4-methylpiperazin-1-yl)methyl-4-dedimethylamino-6-demethyl-6-deoxy-9-hexanoylaminotetracycline

An aqueous solution of 49 mg 37% formaldehyde (0.60 mmol) was added to asolution of 146 mg (0.30 mmol) of4-dedimethylamino-6-demethyl-6-deoxy-9-hexanoylaminotetracycline in 5.0ml ethylene glycol dimethyl ether. The mixture was stirred at roomtemperature for 0.5 hours. 60 mg (0.60 mmol) of 1-methylpiperazine wasthen added and the resulting mixture was stirred overnight and refluxedfor 20 minutes. The mixture was then cooled and a solid product wascollected by filtration. The solid product was then washed with thesolvent and dried by vacuum filtration.

Example 364-Dedimethylamino-6-demethyl-6-deoxy-9-hexanoylaminotetracycline

1.54 g (7.2 mmol) of hexanoic anhydride and 150 mg of 10% Pd/C catalystwere added to 300 mg (0.72 mmol) of4-dedimethylamino-6-demethyl-6-deoxytetracycline in 6.0 ml of1,4-dioxane and 6.0 ml of methanol. The mixture was hydrogenatedovernight at room temperature. The catalyst was removed by filtrationand the filtrate was concentrated under reduced pressure. The residuewas dissolved in 7 ml of ethyl acetate and trituated with 50 ml ofhexane to produce a solid product. The solid product was filtered anddried by vacuum filtration.

Example 37 Phototoxicity Determination

BALB/c 3T3 (CCL-163) cells were obtained from ATCC and cultured inantibiotic-free Dulbecco's Minimum Essential Medium (4.5 g/lglucose)(DMEM) supplemented with L-glutamine (4 mM) and 10% newborn calfserum. The working cell bank was prepared and found to be free ofmycoplasma. Streptomycin sulfate (100 g/ml) and penicillin (100 IU/ml)were added to the medium after the cells were treated with test articlein 96-well plates.

Serial dilutions of the tetracycline derivatives were prepared in DMSOat concentrations 100× to final testing concentration. The COL dilutionsin DMSO were then diluted in Hanks' Balanced Salt Solution (HBSS) forapplication to the cells. The final DMSO concentration was 1% in treatedand control cultures. A dose range finding assay is conducted with eightserial dilutions covering a range of 100-0.03 μg/ml in half log steps.Definitive assays are conducted with 6-8 serial dilutions prepared inquarter log steps, centered on the expected 50% toxicity point asdetermined in the dose range finding assay. One hundred 100 μg/ml wasthe highest dose recommended to prevent false negative results from UVabsorption by the dosing solutions. One dose range finding and at leasttwo definitive trials were performed on each tetracycline derivative andcontrol compound.

Controls: Each assay included both negative (solvent) and positivecontrols. Twelve wells of negative control cultures were used on each96-well plate. Chlorpromazine (Sigma Chemicals) was used as the positivecontrol and was prepared and dosed like the test tetracyclinederivatives.

Solar Simulator: A Dermalight SOL 3 solar simulator, equipped with a UVAH1 filter (320-400 nm), was adjusted to the appropriate height.Measurement of energy through the lid of a 96-well microtiter plate wascarried out using a calibrated UV radiometer UVA sensor. Simulatorheight was adjusted to deliver 1.7±0.1 mW/cm² of UVA energy (resultingdose was 1 J/cm² per 10 minutes of exposure).

Phototoxicity Assay: Duplicate plates were prepared for each testmaterial by seeding 10⁴ 3T3 cells per well in complete medium 24 hoursbefore treatment. Prior to treatment, the medium was removed, and thecells washed once with 125 μl of prewarmed HBSS. Fifty μl of prewarmedHBSS were added to each well. Fifty μl of each test article dilutionwere added to the appropriate wells and the plates returned to theincubator for approximately one hour. Six wells were treated with eachdose of test or control article on each plate. Following the 1 hrincubation, the plates designated for the photo irradiation were exposed(with the lid on) to 1.7±0.1 mW/cm² UVA light for 50±2 minutes at roomtemperature resulting in an irradiation dose of 5 J/cm². Duplicateplates, designated for the measurement of cytotoxicity without light,were kept in the dark room temperature for 50±2 minutes. After the 50minute exposure period (with or without light) the test articledilutions were decanted from the plates and the cells washed once with125 μl of HBSS. One hundred μl of medium were added to all wells and thecells incubated as above for 24±1 hours.

After 24 hours of incubation, the medium was decanted and 100 μl of theNeutral Red containing medium were added to each well. The plates werereturned to the incubator and incubated for approximately 3 hours. After3 hours, the medium was decanted and each well rinsed once with 250 μlof HBSS. The plates were blotted to remove the HBSS and 100 μl ofNeutral Red Solvent were added to each well. After a minimum of 20minutes of incubation at room temperature (with shaking), the absorbanceat 550 nm was measured with a plate reader, using the mean of the blankouter wells as the reference. Relative survival was obtained bycomparing the amount of neutral red taken by each well treated with thetest article and positive control to the neutral red taken up by theaverage of the negative wells (12 wells) on the same plate. The amountof neutral red taken up by the negative control wells is considered tobe 100% survival.

There are several methods by which to quantify relative phototoxicity,e.g., the photoirritancy factor (PIF) and the mean photo effect (MPE),as discussed below.

Phototoxicity Determined by PIF Valuations

To determine the dose where there is a 50% decrease in relativeviability, the relative cell viability is plotted as a function ofincreasing dose and a polynomial equation is calculated to produce the“best fit” line through all the points. The dose of a test substancecorresponding to the point where this line crosses the 50% survivalpoint is calculated (termed the Inhibitory Concentration 50% or IC₅₀)and used to compare the toxicity of the test chemical in the presenceand absence of UVA/visible light.

Phototoxicity of a tetracycline derivative can be measured by itsphotoirritancy factor (PIF). The photo-irritancy factor (PIF) is theratio of the IC₅₀, value in the absence of light to the IC₅₀ value inthe presence of light. That is, the PIF was determined by comparing theIC₅₀ without UVA [IC₅₀(−UVA)] with the IC₅₀ with UVA [IC₅₀(+UVA)]:

${P\; I\; F} = \frac{{IC}_{50}\left( {{- U}\; V\; A} \right)}{{IC}_{50}\left( {{+ U}\; V\; A} \right)}$

IC₅₀ values for both the UVA exposed and non-exposed groups weredetermined whenever possible. If the two values are the same, the PIF is1 and there is no phototoxic effect. If the action of the lightincreases toxicity, the IC₅₀ with light will be lower than the IC₅₀without light, and the PIF will increase.

If IC₅₀ (+UVA) can be determined but IC₅₀(−UVA) cannot, the PIF cannotbe calculated, although the compound tested may have some level ofphototoxic potential. In this case, a “>PIF” can be calculated and thehighest testable dose (−UVA) will be used for calculation of the “>PIF.”

${> {P\; I\; F}} = \frac{{maximum}\mspace{14mu} {{dose}\left( {{- U}\; V\; A} \right)}}{{IC}_{50}\left( {{+ U}\; V\; A} \right)}$

If both, IC₅₀(−UVA) and IC₅₀(+UVA) cannot be calculated because thechemical does not show cytotoxicty (50% reduction in viability) up tothe highest dose tested, this would indicate a lack of phototoxicpotential.

In calculating PIF values, the data resulting from the assay procedurecan be interpreted by different methods.

For example, during the period Mar. 2, 1999 to Apr. 16, 1999, PIF valueswere obtained using the earlier phototoxicity software and itscurve-fitting algorithms, i.e. PIF1.

Since April 1999, the 3T3 phototoxicity assay has undergone extensivevalidation, and has now been incorporated into a draft guideline by theOrganization of Economic Cooperation and Development (OECD) (DraftGuideline 432). (See Spielmann et al., The International EU/COLIPA InVitro Phototoxicity Validation Study; Results of Phase II (blind trial).Part 1: The 3T3 NRU Phototoxicity Test. Toxicology In Vitro 12:305-327(1998); and Spielmann et al., A Study on UV Filter Chemicals from AnnexVII of European Union Directive 76/768/EEC, in the In Vitro 3T3Phototoxicity Test. ATLA 26:679-708 (1998).) The new guideline followsthe same assay procedure, but provides some additional guidance in theinterpretation of the resulting data, and incorporates updated software.As used herein, the PIF value interpreted by this method is termed PIF2.

According to this updated OECD draft guideline, the IC₅₀ values aredeveloped from curves fitted to the data by a multiple boot strapalgorithm. The curve fitting and calculations of the PIF are performedby software developed under contract to the German government (ZEBET,Berlin).

In particular, since there are six wells (and therefore six relativesurvival values) for each dose, the software performs multiplecalculations of the best fit line using what is called boot strapping.This approach is used to account for variations in the data. From thebootstrapped curves, the software determines a mean IC₅₀ for thetreatment. The IC₅₀ is used to compare the toxicity of the test chemicalin the presence and absence of UVA/visible light. FIG. 2 shows anexample of a set of dose response curves prepared for the positivecontrol chemical Chlorpromazine. The difference in the IC₅₀ values canbe clearly seen in this example of a highly phototoxic chemical.

Using the original software and evaluation procedures, if both IC₅₀values can be determined, the cut off value of the factor todiscriminate between phototoxicants and non-phototoxicants is a factorof 5. A factor greater than 5 is indicative of phototoxic potential ofthe test material. Using this software, the mean PIF1 for COL 10 wasdetermined to be 1.83. The mean PIF1 for COL 1002 was determined to be1.12.

The OECD draft guideline has revised the values for the PIF used todifferentiate between phototoxins, potential phototoxins andnon-phototoxins. A PIF2 of less than 2 is considered non-phototoxic, 2to less than 5 is considered potentially phototoxic, and 5 or greater isconsidered clearly phototoxic. In accordance with the OECD draftguideline, the mean PIF2 values of COL 10 and COL 1002 are 2.04 and1.35, respectively.

Phototoxicity Determined by MPE Valuations

At each data point, a photo effect is calculated according to thefollowing formula:

Photo Effect=Dose Effect×Response Effect (i.e., PE _(c) =DE _(c) ×RE_(c))

where c represents one concentration

Dose Effect compares the dose required to achieve percent survival nwithout UVA (c) with the dose required to achieve the same percentsurvival with UVA (c′):

${{Dose}\mspace{14mu} {Effect}_{n}} = \frac{\left( \frac{{Dose}\mspace{14mu} \left( {{- U}\; V\; A} \right)\mspace{14mu} {to}\mspace{14mu} {give}\mspace{14mu} {survival}\mspace{14mu} n}{{Dose}\mspace{14mu} \left( {{+ U}\; V\; A} \right)\mspace{14mu} {to}\mspace{14mu} {give}\mspace{14mu} {survival}\mspace{14mu} n} \right) - 1}{\left( \frac{{Dose}\mspace{14mu} \left( {{- U}\; V\; A} \right)\mspace{14mu} {to}\mspace{14mu} {give}\mspace{14mu} {survival}\mspace{14mu} n}{{Dose}\mspace{14mu} \left( {{+ U}\; V\; A} \right)\mspace{14mu} {to}\mspace{14mu} {give}\mspace{14mu} {survival}\mspace{14mu} n} \right) + 1}$

As the ratio increases, the Dose Effect term approaches 1.

In the example in FIG. 3, the Dose Effect is calculated for one point.The dose of 0.4 dose units is required to reduce cell viability (termedresponse on the y axis) to 66% in the absence of light while only 0.16dose units are required to similarly reduce viability in the presence oflight. The dose effect for 0.4 dose units is:

${DR}_{0,4} = {\frac{{\left( {0.4/0.16} \right) - 1}}{{\left( {0.4/0.16} \right) + 1}} = 0.43}$

The Response Effect at dose c compares the percent survival with andwithout UVA at that dose and normalizes for the total range of theresponse over the range of doses evaluated (n₁ to n_(i)).

${{Response}\mspace{14mu} {Effect}_{c}} = \frac{{{R\left( {{- U}\; V\; A} \right)}c} - {{R\left( {{+ U}\; V\; A} \right)}c}}{R_{0}}$

where R₀ is the Total Survival Range (up to 100%), R(−UVA)c is thesurvival without UVA at dose c, and R(+UVA)c is the survival with UVA atdose c.

As the difference between the survival without UVA at dose c and thesurvival with UVA at dose c [ie., R(−UVA)c−R(+UVA)c] increases(indicative of phototoxic potential), then the Response Effectapproaches 1.0.

Again in FIG. 3, the Response Effect for the 0.4 dose is:

RE _(0.4)=(66%−11%)/100%=0.55

The PE in this example is PE_(0.4)=0.43 *0.55=0.24

The Mean Photo Effect is the mean of the individual Photo Effect valuesover the range evaluated. It is produced from the formula:

${M\; P\; E} = \frac{\sum\limits_{i = 1}^{n}{w_{i}*{PE}_{ci}}}{\sum\limits_{i = 1}^{n}w_{i}}$

where w_(i) is a weighting factor for the highest viability observed foreach curve.

The MPE value is used to determine phototoxic potential. In the originalanalysis of the validation data, a material was considered nonphototoxicif the MPE was <0.1 (this includes negative MPE values) and phototoxicif the MPE was 0.1 (Spielmann et al, 1998). This cut off was re-examinedonce the software had been rewritten and the weighting factor added. Inthe draft Organization for Economic Cooperation and Developmentphototoxicity test guideline (Guideline 432), MPE values of <0.1(including negative values) are considered indicative of anonphototoxin, values of 0.1 to <0.15 are considered probablephototoxins, and greater than and equal to 0.15 clear phototoxins. Thisguideline is expected to become the standard after final approval in2003. The software used to calculate the MPE values is part of thisguideline.

The following table shows the phototoxicity values for severaltetracycline derivatives. The positive control is chlorpromazine. Thephototoxicity is evaluated in terms of MPE and in terms of PIF using thenew OECD draft guideline.

Phototoxicity Values

COMPOUND MPE PIF 1 PIF 2 Chlorpromazine 0.639 N/D 40.38 Tetracycline0.340 5.38 N/A Doxycycline 0.522 23.37 26.71 Minocycline 0.041 2.04 N/ACOL 10 0.099 1.82 2.04 COL 1 0.460 N/D N/A COL 2 0.005 N/D N/A COL 30.654 647 84.72 COL 302 0.378 23.16 23.32 COL 303 0.309 5.27 13.82 COL305 0.420 N/D N/A COL 306 0.038 1.64 1.56 COL 307 0.056 1.17 N/A COL 3080.015 1.0 N/A COL 309 0.170 5.17 12.87 COL 311 0.013 1.0 N/A COL 3120.442 62.67 75.11 COL 313 0.462 80.27 58.22 COL 314 0.475 41.1 89.48 COL315 0.276 15.8 35.30 COL 4 0.570 N/D N/A COL 5 0.186 N/D N/A COL 6 0.155N/D N/A COL 7 0.531 N/D N/A COL 8 0.703 165 82.61 COL 801 −0.001 1.0 N/ACOL 802 −0.123 1.0 N/A COL 803 0.047 N/D N/A COL 804 0.003 1.0 N/A COL805 0.022 1.0 N/A COL 807 0.382 40.4 N/A COL 808 0.387 46.45 N/A COL 8090.420 N/D N/A COL 9 0.546 N/D N/A COL 1001 0.025 N/D N/A COL 1002 0.0401.0 1.35 N/A indicates that the IC₅₀ value could not be determined forthe UVA exposed and/or non-exposed groups N/D indicates that the PIF 1was not determined for the particular compound, or was N/A as definedabove.

In the present specification, some of the compounds of the invention arereferred to by codes names. The correspondence between the compound andcodes names are as follows:

Chemical Names of the COL Compounds

COL-1 4-dedimethylaminotetracycline COL-36-demethyl-6-deoxy-4-dedimethylaminotetracycline COL-3017-bromo-6-demethyl-6-deoxy- 4-dedimethylaminotetracycline COL-3027-nitro-6-demethyl-6-deoxy- 4-dedimethylaminotetracycline COL-3039-nitro-6-demethyl-6-deoxy- 4-dedimethylaminotetracycline COL-3047-acetamido-6-demethyl-6-deoxy- 4-dedimethylaminotetracycline COL-3059-acetamido-6-demethyl-6-deoxy- 4-dedimethylaminotetracycline COL-3069-dimethylamino-6-demethyl-6-deoxy- 4-dedimethylaminotetracyclineCOL-307 7-amino-6-demethyl-6-deoxy- 4-dedimethylaminotetracyclineCOL-308 9-amino-6-demethyl-6-deoxy- 4-dedimethylaminotetracyclineCOL-309 9-dimethylaminoacetamido-6-demethyl-6-deoxy-4-dedimethylaminotetracycline COL-3107-dimethylamino-6-demethyl-6-deoxy- 4-dedimethylaminotetracyclineCOL-311 9-palmitamide-6-demethyl-6-deoxy- 4-dedimethylaminotetracyclineCOL-312 2-CONHCH₂-pyrrolidin-1-yl-6-demethyl-6-deoxy-4-dedimethylaminotetracycline COL-3132-CONHCH₂-piperidin-1-yl-6-demethyl-6-deoxy-4-dedimethylaminotetracycline COL-3142-CONHCH₂-morpholin-1-yl-6-demethyl-6-deoxy-4-dedimethylaminotetracycline COL-3152-CONHCH₂-piperazin-1-yl-6-demethyl-6-deoxy-4-dedimethylaminotetracycline COL-47-chloro-4-dedimethylaminotetracycline COL-5 tetracycline pyrazole COL-64-hydroxy-4-dedimethylaminotetracycline COL-74-dedimethylamino-12α-deoxytetracycline COL-84-dedimethylaminodoxycycline COL-8019-acetamido-4-dedimethylaminodoxycycline COL-8029-dimethylaminoacetamido- 4-dedimethylaminodoxycycline COL-8039-palmitamide-4-dedimethylaminodoxycycline COL-8049-nitro-4-dedimethylaminodoxycycline COL-8059-amino-4-dedimethylaminodoxycycline COL-8069-dimethylamino-4-dedimethylaminodoxycycline COL-8072-CONHCH₂-pyrrolidin-1-yl- 4-dedimethylaminodoxycycline COL-8082-CONHCH₂-piperidin-1-yl- 4-dedimethylaminodoxycycline COL-8092-CONHCH₂-piperazin-1-yl- 4-dedimethylaminodoxycycline COL-104-dedimethylaminominocycline (a.k.a. COL-310) COL-10017-trimethylammonium-4-dedimethylaminosancycline COL-10029-nitro-4-dedimethylaminominocycline

Index of Structures

wherein R7 is selected from the group consisting of hydrogen, amino,nitro, mono(lower alkyl)amino, halogen, di(lower alkyl)amino,ethoxythiocarbonylthio, azido, acylamino, diazonium, cyano, andhydroxyl; R6-a is selected from the group consisting of hydrogen andmethyl; R6 and R5 are selected from the group consisting of hydrogen andhydroxyl; R8 is selected from the group consisting of hydrogen andhalogen; R9 is selected from the group consisting of hydrogen, amino,azido, nitro, acylamino, hydroxy, ethoxythiocarbonylthio, mono(loweralkyl)amino, halogen, diazonium, di(lower alkyl)amino and RCH(NH₂)CO; Ris hydrogen or lower alkyl; and pharmaceutically acceptable andunacceptable salts thereof; with the following provisos: when either R7and R9 are hydrogen then R8 must be halogen; and when R6-a, R6, R5 andR9 are all hydrogen and R7 is hydrogen, amino, nitro, halogen,dimethylamino or diethylamino, then R8 must be halogen; and when R6-a ismethyl, R6 and R9 are both hydrogen, R5 is hydroxyl and R7 is hydrogen,amino, nitro, halogen or diethylamino, then R8 is halogen; and when R6-ais methyl, R6 is hydroxyl, R5, R7 and R9 are all hydrogen, then R8 mustbe halogen; and when R6-a, R6 and R5 are all hydrogen, R9 is methylaminoand R7 is dimethylamino, then R8 must be halogen; and when R6-a ismethyl, R6 is hydrogen, R5 is hydroxyl, R9 is methylamino and R7 isdimethylamino, then R8 must be halogen; and when R6-a is methyl, R6, R5and R9 are all hydrogen and R7 is cyano, then R8 must be halogen.

wherein R7 is selected from the group consisting of hydrogen, amino,nitro, mono(lower alkyl)amino, halogen, di(lower alkyl)amino,ethoxythiocarbonylthio, azido, acylamino, diazonium, cyano, andhydroxyl; R6-a is selected from the group consisting of hydrogen andmethyl; R6 and R5 are selected from the group consisting of hydrogen andhydroxyl; R4 is selected from the group consisting of NOH, N—NH-A, andNH-A, where A is a lower alkyl group; R8 is selected from the groupconsisting of hydrogen and halogen; R9 is selected from the groupconsisting of hydrogen, amino, azido, nitro, acylamino, hydroxy,ethoxythiocarbonylthio, mono(lower alkyl)amino, halogen, di(loweralkyl)amino and RCH(NH₂)CO; R is hydrogen or lower alkyl; andpharmaceutically acceptable and unacceptable salts thereof; with thefollowing provisos: when R4 is NOH, N—NH-alkyl or NH-alkyl and R7, R6-a,R6, R5, and R9 are all hydrogen, then R8 must be halogen; and when R4 isNOH, R6-a is methyl, R6 is hydrogen or hydroxyl, R7 is halogen, R5 andR9 are both hydrogen, then R8 must be halogen; and when R4 isN—NH-alkyl, R6-a is methyl, R6 is hydroxyl and R7, R5, R9 are allhydrogen, then R8 must be halogen; and when R4 is NH-alkyl, R6-a, R6, R5and R9 are all hydrogen, R7 is hydrogen, amino, mono(lower alkyl)amino,halogen, di(lower alkyl)amino or hydroxyl, then R8 must be halogen; andwhen R4 is NH-alkyl, R6-a is methyl, R6 and R9 are both hydrogen, R5 ishydroxyl, and R7 is mono(lower alkyl)amino or di(lower alkyl)amino, thenR8 must be halogen; and when R4 is NH-alkyl, R6-a is methyl, R6 ishydroxy or hydrogen and R7, R5, and R9 are all be hydrogen, then R8 mustbe halogen.

wherein R7, R8, and R9 taken together in each case, have the followingmeanings:

R7 R8 R9 azido hydrogen hydrogen dimethylamino hydrogen azido hydrogenhydrogen amino hydrogen hydrogen azido hydrogen hydrogen nitrodimethylamino hydrogen amino acylamino hydrogen hydrogen hydrogenhydrogen acylamino amino hydrogen nitro hydrogen hydrogen(N,N-dimethyl)glycylamino amino hydrogen amino hydrogen hydrogenethoxythiocarbonylthio dimethylamino hydrogen acylamino dimethylaminohydrogen diazonium dimethylamino chloro amino hydrogen chloro aminoamino chloro amino acylamino chloro acylamino amino chloro hydrogenacylamino chloro hydrogen monoalkylamino chloro amino nitro chloro aminodimethylamino chloro acylamino dimethylamino chloro dimethylaminodimethylamino hydrogen hydrogen hydrogen hydrogen dimethylaminotrimethylammonium hydrogen hydrogenand

wherein R7, R8, and R9 taken together in each case, have the followingmeanings:

R7 R8 R9 azido hydrogen hydrogen dimethylamino hydrogen azido hydrogenhydrogen amino hydrogen hydrogen azido hydrogen hydrogen nitrodimethylamino hydrogen amino acylamino hydrogen hydrogen hydrogenhydrogen acylamino amino hydrogen nitro hydrogen hydrogen(N,N-dimethyl)glycylamino amino hydrogen amino hydrogen hydrogenethoxythiocarbonylthio dimethylamino hydrogen acylamino hydrogenhydrogen diazonium hydrogen hydrogen dimethylamino diazonium hydrogenhydrogen ethoxythiocarbonylthio hydrogen hydrogen dimethylamino chloroamino amino chloro amino acylamino chloro acylamino hydrogen chloroamino amino chloro hydrogen acylamino chloro hydrogen monoalkyl aminochloro amino nitro chloro aminoand

wherein R8 is hydrogen or halogen and R9 is selected from the groupconsisting of nitro, (N,N-dimethyl)glycylamino, andethoxythiocarbonylthio; and

wherein R7, R8, and R9 taken together in each case, have the followingmeanings:

R7 R8 R9 amino hydrogen hydrogen nitro hydrogen hydrogen azido hydrogenhydrogen dimethylamino hydrogen azido hydrogen hydrogen amino hydrogenhydrogen azido hydrogen hydrogen nitro bromo hydrogen hydrogendimethylamino hydrogen amino acylamino hydrogen hydrogen hydrogenhydrogen acylamino amino hydrogen nitro hydrogen hydrogen(N,N-dimethyl)glycylamino amino hydrogen amino diethylamino hydrogenhydrogen hydrogen hydrogen ethoxythiocarbonylthio dimethylamino hydrogenmethylamino dimethylamino hydrogen acylamino dimethylamino chloro aminoamino chloro amino acylamino chloro acylamino hydrogen chloro aminoamino chloro hydrogen acylamino chloro hydrogen monoalkylamino chloroamino nitro chloro aminoand pharmaceutically acceptable and unacceptable salts thereof

wherein R7 is selected from the group consisting of hydrogen, amino,nitro, mono(lower alkyl)amino, halogen, di(lower alkyl)amino,ethoxythiocarbonylthio, azido, acylamino, diazonium, cyano, andhydroxyl; R6-a is selected from the group consisting of hydrogen andmethyl; R6 and R5 are selected from the group consisting of hydrogen andhydroxyl; R8 is selected from the group consisting of hydrogen andhalogen; R9 is selected from the group consisting of hydrogen, amino,azido, nitro, acylamino, hydroxy, ethoxythiocarbonylthio, mono(loweralkyl amino, halogen, diazonium, di(lower alkyl)amino and RCH(NH₂)CO; Ris hydrogen or lower alkyl; R^(a) and R^(b) are selected from the groupconsisting of hydrogen, methyl, ethyl, n-propyl and 1-methylethyl withthe proviso that R^(a) and R^(b) cannot both be hydrogen; R^(e) andR^(d) are, independently (CH₂)_(n)CHR^(e) wherein n is 0 or 1 and R^(e)is selected from the group consisting of hydrogen, alkyl, hydroxy, lower(C₁-C₃)alkoxy, amino, or nitro; and, W is selected from the groupconsisting of (CHR^(e))_(m), wherein m is 0-3 and R^(e) is as above, NH,N(C₁-C₃) straight chained or branched alkyl, O, S and N(C₁-C₄) straightchain or branched alkoxy; and pharmaceutically acceptable andunacceptable salts thereof. In a further embodiment, the followingprovisos apply: when either R⁷ and R⁹ are hydrogen then R8 must behalogen; and when R6-a, R6, R5 and R9 are all hydrogen and R7 ishydrogen, amino, nitro, halogen, dimethylamino or diethylamino, then R8must be halogen; and when R6-a is methyl, R6 and R9 are both hydrogen,R5 is hydroxyl, and R7 is hydrogen, amino, nitro, halogen ordiethylamino, then R8 is halogen; and when R6-a is methyl, R6 ishydroxyl, R5, R7 and R9 are all hydrogen, then R8 must be halogen; andwhen R6-a, R6 and R5 are all hydrogen, R9 is methylamino and R7 isdimethylamino, then R8 must be halogen; and when R6-a is methyl, R6 ishydrogen, R5 is hydroxyl, R9 is methylamino and R7 is dimethylamino,then R8 must be halogen; and when R6-a is methyl, R6, R5 and R9 are allhydrogen and R7 is cyano, then R8 must be halogen.

Structure K

wherein: R7, R8, and R9 taken together in each case, have the followingmeanings:

R7 R8 R9 hydrogen hydrogen amino hydrogen hydrogen palmitamideand

Structure L Structure M Structure N Structure O

wherein: R7, R8, and R9 taken together in each case, have the followingmeanings:

R7 R8 R9 hydrogen hydrogen acetamido hydrogen hydrogendimethylaminoacetamido hydrogen hydrogen nitro hydrogen hydrogen aminoand

Structure P

wherein: R8, and R9 taken together are, respectively, hydrogen andnitro.

Structure K

wherein: R7, R8, and R9 taken together are, respectively, hydrogen,hydrogen and dimethylamino.

STRUCTURE C STRUCTURE D STRUCTURE E STRUCTURE Fwherein R7 is selected from the group consisting of an aryl, alkenyl andalkynyl; R6-a is selected from the group consisting of hydrogen andmethyl; R6 and R5 are selected from the group consisting of hydrogen andhydroxyl; R8 is selected from the group consisting of hydrogen andhalogen; R9 is selected from the group consisting of hydrogen, amino,azido, nitro, acylamino, hydroxy, ethoxythiocarbonylthio, mono(loweralkyl)amino, halogen, diazonium, di(lower alkyl)amino and RCH(NH₂)CO;and pharmaceutically acceptable and unacceptable salts thereof

or

STRUCTURE C STRUCTURE D STRUCTURE E STRUCTURE Fwherein: R7 is selected from the group consisting of hydrogen, amino,nitro, mono(lower alkyl)amino, halogen, di(lower alkyl)amino,ethoxythiocarbonylthio, azido, acylamino, diazonium, cyano, andhydroxyl; R6-a is selected from the group consisting of hydrogen andmethyl; R6 and R5 are selected from the group consisting of hydrogen andhydroxyl; R8 is selected from the group consisting of hydrogen andhalogen; R9 is selected from the group consisting of an aryl, alkenyland alkynyl; and pharmaceutically acceptable and unacceptable saltsthereof;or

STRUCTURE C STRUCTURE D STUCTURE E STRUCTURE Fwherein: R7 and R9 are selected from the group consisting of an aryl,alkene, alkyne, or mixtures thereof; R6-a is selected from the groupconsisting of hydrogen and methyl; R6 and R5 are selected from the groupconsisting of hydrogen and hydroxyl; R8 is selected from the groupconsisting of hydrogen and halogen; and pharmaceutically acceptable andunacceptable salts thereof

STRUCTURE G STRUCTURE H STRUCTURE I STRUCTURE Jwherein R7 is selected from the group consisting of an aryl, alkenyl andalkynyl; R6-a is selected from the group consisting of hydrogen andmethyl; R6 and R5 are selected from the group consisting of hydrogen andhydroxyl; R4 is selected from the group consisting of NOH, N—NH-A, andNH-A, where A is a lower alkyl group; R8 is selected from the groupconsisting of hydrogen and halogen; R9 is selected from the groupconsisting of hydrogen, amino, azido, nitro, acylamino, hydroxy,ethoxythiocarbonylthio, mono(lower alkyl)amino, halogen, di(loweralkyl)amino and RCH(NH₂)CO; and pharmaceutically acceptable andunacceptable salts thereof;or

STRUCTURE G STRUCTURE H STRUCTURE I STRUCTURE Jwherein R7 is selected from the group consisting of hydrogen, amino,nitro, mono(lower alkyl)amino, halogen, di(lower alkyl)amino,ethoxythiocarbonylthio, azido, acylamino, diazonium, cyano, andhydroxyl; R6-a is selected from the group consisting of hydrogen andmethyl; R6 and R5 are selected from the group consisting of hydrogen andhydroxyl; R4 is selected from the group consisting of NOH, N—NH-A, andNH-A, where A is a lower alkyl group; R8 is selected from the groupconsisting of hydrogen and halogen; R9 is selected from the groupconsisting of an aryl, alkenyl and alkynyl; and pharmaceuticallyacceptable and unacceptable salts thereof;or

STRUCTURE G STRUCTURE H STRUCTURE I STRUCTURE Jwherein: R7 and R9 are selected from the group consisting of an aryl,alkenyl, alkynyl; or mixtures thereof; R6-a is selected from the groupconsisting of hydrogen and methyl; R6 and R5 are selected from the groupconsisting of hydrogen and hydroxyl; R4 is selected from the groupconsisting of NOH, N—NH-A, and NH-A, where A is a lower alkyl group; andR8 is selected from the group consisting of hydrogen and halogen; andpharmaceutically acceptable and unacceptable salts thereof.

Structure K

wherein R7 is selected from the group consisting of an aryl, alkenyl andalkynyl; R8 is selected from the group consisting of hydrogen andhalogen; R9 is selected from the group consisting of hydrogen, amino,azido, nitro, acylamino, hydroxy, ethoxythiocarbonylthio, mono(loweralkyl)amino, halogen, di(lower alkyl)amino and RCH(NH₂)CO; andpharmaceutically acceptable and unacceptable salts thereof;

or

Structure K

wherein: R7 is selected from the group consisting of hydrogen, amino,nitro, mono(lower alkyl)amino, halogen, di(lower alkyl)amino,ethoxythiocarbonylthio, azido, acylamino, diazonium, cyano, andhydroxyl; R8 is selected from the group consisting of hydrogen andhalogen; R9 is selected from the group consisting of an aryl, alkenyland alkynyl; and pharmaceutically acceptable and unacceptable saltsthereof;

or

Structure K

wherein: R7 and R9 are selected from the group consisting of an aryl,alkenyl, alkynyl and mixtures thereof; and R8 is selected from the groupconsisting of hydrogen and halogen; and pharmaceutically acceptable andunacceptable salts thereof; and

STRUCTURE L STRUCTURE M STRUCTURE N STRUCTURE Owherein: R7 is selected from the group consisting of an aryl, alkenyland alkynyl; R8 is selected from the group consisting of hydrogen andhalogen; and pharmaceutically acceptable and unacceptable salts thereof;or

STRUCTURE L STRUCTURE M STRUCTURE N STRUCTURE Owherein R7 is selected from the group consisting of hydrogen, amino,nitro, mono(lower alkyl)amino, halogen, di(lower alkyl)amino,ethoxythiocarbonylthio, azido, acylamino, diazonium, cyano, andhydroxyl; R8 is selected from the group consisting of hydrogen andhalogen; R9 is selected from the group consisting of an aryl, alkenyland alkynyl; and pharmaceutically acceptable and unacceptable saltsthereof;or

STRUCTURE L STRUCTURE M STRUCTURE N STRUCTURE Owherein R7 is and R9 are selected from the group consisting of an aryl,alkenyl, alkynyl and mixtures thereof; R8 is selected from the groupconsisting of hydrogen and halogen; R9 is selected from the groupconsisting of hydrogen, amino, azido, nitro, acylamino, hydroxy,ethoxythiocarbonylthio, mono(lower alkyl)amino, halogen, di(loweralkyl)amino and RCH(NH₂)CO; and pharmaceutically acceptable andunacceptable salts thereof;and

Structure P

wherein R9 is selected from the group consisting of an aryl, alkenyl andalkynyl; and R8 is selected from the group consisting of hydrogen andhalogen; and pharmaceutically acceptable and unacceptable salts thereof;

and

STRUCTURE Q STRUCTURE Rwherein R7 is selected from the group consisting of an aryl, alkenyl andalkynyl; R8 is selected from the group consisting of hydrogen andhalogen; R9 is selected from the group consisting of hydrogen, amino,azido, nitro, acylamino, hydroxy, ethoxythiocarbonylthio, mono(loweralkyl)amino, halogen, di(lower alkyl)amino and RCH(NH₂)CO; andpharmaceutically acceptable and unacceptable salts thereof;or

STRUCTURE Q STRUCTURE Rwherein R7 is selected from the group consisting of hydrogen, amino,nitro, mono(lower alkyl)amino, halogen, di(lower alkyl)amino,ethoxythiocarbonylthio, azido, acylamino, diazonium, cyano, andhydroxyl; R8 is selected from the group consisting of hydrogen andhalogen; R9 is selected from the group consisting of an aryl, alkenyland alkynyl; and pharmaceutically acceptable and unacceptable saltsthereof;or

STRUCTURE Q STRUCTURE Rwherein R7 and R9 are selected from the group consisting of an aryl,alkenyl, alkynyl; and mixtures thereof; R8 is selected from the groupconsisting of hydrogen and halogen; and pharmaceutically acceptable andunacceptable salts thereof.

Structures S—Z

wherein R7 is selected from the group consisting of an aryl, alkenyl andalkynyl; R6-a is selected from the group consisting of hydrogen andmethyl; R6 and R5 are selected from the group consisting of hydrogen andhydroxyl; R8 is selected from the group consisting of hydrogen andhalogen; R9 is selected from the group consisting of hydrogen, amino,azido, nitro, acylamino, hydroxy, ethoxythiocarbonylthio, mono(loweralkyl)amino, halogen, diazonium, di(lower alkyl)amino and RCH(NH₂)CO;R^(a) and R^(b) are selected from the group consisting of hydrogen,methyl, ethyl, n-propyl and 1-methylethyl with the proviso that R^(a)and R^(b) cannot both be hydrogen; R^(e) and R^(d) are, independently,(CH₂)_(n)CHR^(e) wherein n is 0 or 1 and R^(e) is selected from thegroup consisting of hydrogen, alkyl, hydroxy, lower (C₁-C₃)alkoxy,amino, or nitro; and, W is selected from the group consisting of(CHR^(e))_(m) wherein m is 0-3 and said R^(e) is as above, NH, N(C₁-C₃)straight chained or branched alkyl, O, S and N(C₁-C₄) straight chain orbranched alkoxy; and pharmaceutically acceptable and unacceptable saltsthereof;

or

Structures S—Z

wherein R7 is selected from the group consisting of hydrogen, amino,nitro, mono(lower alkyl)amino, halogen, di(lower alkyl)amino,ethoxythiocarbonylthio, azido, acylamino, diazonium, cyano, andhydroxyl; R6-a is selected from the group consisting of hydrogen andmethyl; R6 and R5 are selected from the group consisting of hydrogen andhydroxyl; R8 is selected from the group consisting of hydrogen andhalogen; R9 is selected from the group consisting of an aryl, alkenyland alkynyl; R^(a) and R^(b) are selected from the group consisting ofhydrogen, methyl, ethyl, n-propyl and 1-methylethyl with the provisothat R^(a) and R^(b) cannot both be hydrogen; R^(e) and R^(d) are,independently, (CH₂)_(n)CHR^(e) wherein n is 0 or 1 and R^(e) isselected from the group consisting of hydrogen, alkyl, hydroxy, lower(C₁-C₃)alkoxy, amino, or nitro; and, W is selected from the groupconsisting of (CHR^(e))_(m) wherein m is 0-3 and said R^(e) is as above,NH, N(C₁-C₃) straight chained or branched alkyl, O, S and N(C₁-C₄)straight chain or branched alkoxy; and pharmaceutically acceptable andunacceptable salts thereof;

or

Structures S—Z

wherein: R7 and R9 are selected from the group consisting of an aryl,alkenyl, alkynyl and mixtures thereof; R6-a is selected from the groupconsisting of hydrogen and methyl; R6 and R5 are selected from the groupconsisting of hydrogen and hydroxyl; R8 is selected from the groupconsisting of hydrogen and halogen; R^(a) and R^(b) are selected fromthe group consisting of hydrogen, methyl, ethyl, n-propyl and1-methylethyl with the proviso that R^(a) and R^(b) cannot both behydrogen; R^(e) and R^(d) are, independently, (CH₂)_(n)CHR^(e) wherein nis 0 or 1 and R^(e) is selected from the group consisting of hydrogen,alkyl, hydroxy, lower (C₁-C₃)alkoxy, amino, or nitro; and W is selectedfrom the group consisting of (CHR^(e))_(m) wherein m is 0-3 and saidR^(e) is as above, NH, N(C₁-C₃) straight chained or branched alkyl, O, Sand N(C₁-C₄) straight chain or branched alkoxy; and pharmaceuticallyacceptable and unacceptable salts thereof.

Throughout this specification, the descriptions of some structuresinclude the term “lower alkyl.” The term “lower alkyl” means an alkylgroup comprising relatively few carbon atoms, for example, about one toten carbon atoms. A preferred low end of this range is one, two, three,four or five carbon atoms; and a preferred high end of this range issix, seven, eight, nine or ten carbon atoms. Some examples of “loweralkyl” groups include methyl groups, ethyl groups, propyl groups,isopropyl groups, butyl groups, etc.

1. A method for simultaneously treating ocular rosacea and acne rosaceain a human in need thereof comprising administering systemically to saidhuman a tetracycline compound in an amount that is effective to treatocular rosacea and acne rosacea but has substantially no antibioticactivity. 2-45. (canceled)